Bottom Line:
Depletion of Dab2 inhibits cell migration and polarized movement of integrin beta1 and vinculin to the leading edge.By manipulating intracellular and surface integrin beta1 levels, we show that migration speed correlates with the intracellular integrin pool but not the surface level.Together, these results suggest that Dab2 internalizes integrins freely diffusing on the cell surface and that Dab2 regulates migration, perhaps by maintaining an internal pool of integrins that can be recycled to create new adhesions at the leading edge.

ABSTRACTClathrin-associated endocytic adapters recruit cargoes to coated pits as a first step in endocytosis. We developed an unbiased quantitative proteomics approach to identify and quantify glycoprotein cargoes for an endocytic adapter, Dab2. Surface levels of integrins beta1, alpha1, alpha2, and alpha3 but not alpha5 or alphav chains were specifically increased on Dab2-deficient HeLa cells. Dab2 colocalizes with integrin beta1 in coated pits that are dispersed over the cell surface, suggesting that it regulates bulk endocytosis of inactive integrins. Depletion of Dab2 inhibits cell migration and polarized movement of integrin beta1 and vinculin to the leading edge. By manipulating intracellular and surface integrin beta1 levels, we show that migration speed correlates with the intracellular integrin pool but not the surface level. Together, these results suggest that Dab2 internalizes integrins freely diffusing on the cell surface and that Dab2 regulates migration, perhaps by maintaining an internal pool of integrins that can be recycled to create new adhesions at the leading edge.

Mentions:
We used SILAC (stable isotope labeling with amino acids in cell culture) labeling for quantification and identified cell surface–exposed proteins using the recently developed cell surface capture (CSC) technology (Fig. 1 A; Ong et al., 2002; Wollscheid et al., 2009). Dab2-deficient HeLa cells were generated by use of a retroviral vector carrying Dab2-specific short hairpin RNA (shRNA). A control HeLa cell line was generated in parallel using control shRNA (Fig. 1 B). Dab2-deficient cells were grown in SILAC media containing [13C6,15N4]l-arginine and [13C6]l-lysine, and control cells were grown in normal media (Ong et al., 2002). After five to six generations, the cells were harvested and mixed, and their surface glycoproteins were tagged with biocytin hydrazide (Wollscheid et al., 2009). Subsequently, the cells were lysed, and a microsomal pellet was prepared. Microsomal proteins were trypsinized, and tagged glycopeptides were affinity purified with streptavidin beads. N-glycosylated peptides were specifically eluted by using protein N-glycosidase F. Eluted N-glycosites (formerly N-glycosylated peptides) were identified and quantified using mass spectrometry–based proteomics. Of the ∼100–150 surface glycoproteins detected in two independent experiments, 57 were identified, and 41 were reliably quantified in both experiments (Tables S1 and S2). 13 glycoproteins were reproducibly increased on the surface of Dab2-deficient cells (i.e., their light/heavy isotope ratios decreased in both experiments). 5 of these 13 glycoproteins were integrins (Fig. 1 C). Surface levels of integrins β1, α1, α2, and α3 increased the most, and integrin α5 increased slightly. Integrin αv was not changed (Table S2). Because an effect of Dab2 on integrins may partly explain the down-regulation of Dab2 in cancer, we investigated the effect of Dab2 on integrins in detail.

Mentions:
We used SILAC (stable isotope labeling with amino acids in cell culture) labeling for quantification and identified cell surface–exposed proteins using the recently developed cell surface capture (CSC) technology (Fig. 1 A; Ong et al., 2002; Wollscheid et al., 2009). Dab2-deficient HeLa cells were generated by use of a retroviral vector carrying Dab2-specific short hairpin RNA (shRNA). A control HeLa cell line was generated in parallel using control shRNA (Fig. 1 B). Dab2-deficient cells were grown in SILAC media containing [13C6,15N4]l-arginine and [13C6]l-lysine, and control cells were grown in normal media (Ong et al., 2002). After five to six generations, the cells were harvested and mixed, and their surface glycoproteins were tagged with biocytin hydrazide (Wollscheid et al., 2009). Subsequently, the cells were lysed, and a microsomal pellet was prepared. Microsomal proteins were trypsinized, and tagged glycopeptides were affinity purified with streptavidin beads. N-glycosylated peptides were specifically eluted by using protein N-glycosidase F. Eluted N-glycosites (formerly N-glycosylated peptides) were identified and quantified using mass spectrometry–based proteomics. Of the ∼100–150 surface glycoproteins detected in two independent experiments, 57 were identified, and 41 were reliably quantified in both experiments (Tables S1 and S2). 13 glycoproteins were reproducibly increased on the surface of Dab2-deficient cells (i.e., their light/heavy isotope ratios decreased in both experiments). 5 of these 13 glycoproteins were integrins (Fig. 1 C). Surface levels of integrins β1, α1, α2, and α3 increased the most, and integrin α5 increased slightly. Integrin αv was not changed (Table S2). Because an effect of Dab2 on integrins may partly explain the down-regulation of Dab2 in cancer, we investigated the effect of Dab2 on integrins in detail.

Bottom Line:
Depletion of Dab2 inhibits cell migration and polarized movement of integrin beta1 and vinculin to the leading edge.By manipulating intracellular and surface integrin beta1 levels, we show that migration speed correlates with the intracellular integrin pool but not the surface level.Together, these results suggest that Dab2 internalizes integrins freely diffusing on the cell surface and that Dab2 regulates migration, perhaps by maintaining an internal pool of integrins that can be recycled to create new adhesions at the leading edge.

ABSTRACTClathrin-associated endocytic adapters recruit cargoes to coated pits as a first step in endocytosis. We developed an unbiased quantitative proteomics approach to identify and quantify glycoprotein cargoes for an endocytic adapter, Dab2. Surface levels of integrins beta1, alpha1, alpha2, and alpha3 but not alpha5 or alphav chains were specifically increased on Dab2-deficient HeLa cells. Dab2 colocalizes with integrin beta1 in coated pits that are dispersed over the cell surface, suggesting that it regulates bulk endocytosis of inactive integrins. Depletion of Dab2 inhibits cell migration and polarized movement of integrin beta1 and vinculin to the leading edge. By manipulating intracellular and surface integrin beta1 levels, we show that migration speed correlates with the intracellular integrin pool but not the surface level. Together, these results suggest that Dab2 internalizes integrins freely diffusing on the cell surface and that Dab2 regulates migration, perhaps by maintaining an internal pool of integrins that can be recycled to create new adhesions at the leading edge.